Genes and vectors for conferring herbicide resistance in plants

ABSTRACT

Genomic and cDNA sequences and plant expression vectors for encoding an eukaryotic AHAS small subunit are disclosed. The DNA sequences and vectors are used to transform plants to produce transgenic plants which possess elevated levels of tolerance or resistance to herbicides, such as imidazolinones.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.09/426,568, filed Oct. 22, 1999, now U.S. Pat. No. 6,348,643, whichclaims the benefit, under 35 U.S.C. §119(e), of U.S. Provisional PatentApplication Ser. No. 60/106,239, filed Oct. 29, 1998; both of which arehereby incorporated herein in their entirety by reference.

BACKGROUND OF THE INVENTION

Herbicides are used extensively in agronomy for controlling weeds andother undesirable plants. Because of their phytotoxicity, herbicidesalso kill or significantly inhibit the growth and yield of desirableplants.

Some plants, for example Arabidopsis, inherently possess or developresistance to certain herbicides upon repeated exposure to herbicideswith the same mode of action. It has been a goal of plantbiotechnologists to identify, isolate and clone plant genes that conferresistance to herbicides and use these genes to transform desirableplants such as crops to render them herbicide resistant.

Several methods for generating or identifying herbicide resistance inplants are known. For example, U.S. Pat. Nos. 5,719,046, 5,633,444 and5,597,717 disclose a plant sulfonamide resistant gene and methods fortransforming plant cells whose growth is inhibited by sulfonamides, withvectors containing this gene.

U.S. Pat. No. 5,405,765 discloses a method for producing transgenicwheat plants. This method comprises delivering a heterologous DNA to aType C embryonic wheat callus in a suspension culture by an acceleratedparticle bombardment method.

U.S. Pat. No. 5,539,092 discloses polynucleotides encoding acyanobacterial and plant acetyl-CoA carboxylase. This patent disclosesprocesses for increasing the herbicide resistance of a monocotyledonousplant, comprising transforming the plant with a DNA molecule encoding aherbicide resistant polypeptide having the ability to catalyze thecarboxylation of acetyl-CoA. The patent further discloses that thetransgenic plants produced are resistant to herbicides such asarylphenoxyproprionates and cyclohexanediones.

U.S. Pat. No. 5,304,732 discloses methods for isolatingherbicide-resistant plants. The patent describes the use of in vitrocell culture methods for isolating plant cell lines that are resistantto herbicides such as imidazolinones and sulfonamides.

The trait for a specific herbicide resistance is most often associatedwith a particular enzyme. One such enzyme which has been of interest inits association of conferring herbicide resistance in plants isacetohydroxy-acid synthase (“AHAS”), also known as acetolactate synthase(“ALS,” E.C. 4.1.3.18). It is an essential enzyme in plants and manymicroorganisms, and in most plants the enzyme is sensitive toherbicides. The AHAS enzyme catalyzes the first step in the biosynthesisof the branched-chain amino acids, isoleucine, leucine, and valine, andits activity is allosterically inhibited by these amino acids. AHASactivity is also inhibited by several classes of herbicides, includingimidazolinone compounds such as imazethapyr (PURSUIT®, AmericanCyanamid, Parsipanny, N.J.); sulfonylurea-based compounds such assufometuron methyl (OUST®,), E. I. du Pont de Nemours and Company,Wilmington, Del.); triazolopyrimidine sulfonamides (Broadstrike™, DowElanco; see Gerwick et al. Pestic. Sci. 29: 357-364, 1990);sulfamoylureas (Rodaway et al., Mechanism of Selectively of Ac 322,140in Paddy Rice, Wheat and Barley, Proc. Brighton Crop Protec. Conf.,Weeds, 1993); pyrimidyl-oxy-benzoic acids (STABLE®, Kumiai ChemicalIndustry Co., E. I. du Pont de Nemours and Company), andsulfonylcarboxamides (Alvarado et al., U.S. Pat. No. 4,883,914).Inhibition of AHAS activity may lead to the inability of the plant tomake the branched amino acids or to the accumulation of toxicmetabolites and, therefore, plant death.

Genes encoding AHAS enzymes have been isolated from enteric bacteria,including Escherichia coli, and Salmonella typhimurium. U.S. Pat. No.5,643,779 discloses a nucleic acid sequence coding for an α-AHAS enzymefrom Lactococcus and vectors containing same for transformingmicroorganisms. The transgenic microorganisms produce an enhanced amountof the AHAS enzyme.

Japanese Patent Document No. JP08214882 discloses a nucleic acidsequence for a large and a small subunit of AHAS from Rhodobactercapsulatus. The gene sequences are used to transform photosyntheticmicroorganisms to improve the production of AHAS enzyme for thesynthesis of amino acids.

In eukaryotes, a gene encoding a polypeptide homologous to the largesubunits of bacterial AHAS enzymes has been identified in the yeastSaccharomyces cerevisiae. Genes encoding mutant large subunits of AHASfrom various plants have also been isolated, cloned, and used to createtransgenic plants that are resistant to herbicides.

U.S. Pat. Nos. 5,605,011, 5,013,659, 5,141,870 and 5,378,824 disclosenucleic acid fragments encoding a mutant plant ALS protein associatedwith herbicide resistance. The mutant ALS large subunit protein confersherbicide resistance to sulfonylurea compounds in plants. The nucleicacid fragments encoding this mutant large subunit protein are used invectors to transform plants which are normally sensitive to sulfonylureaherbicides. The transgenic plants resulting from such transformation areresistant to sulfonylurea herbicides.

U.S. Pat. No. 5,633,437 discloses a large subunit gene and enzymeisolated from cocklebur, Xanthium sp., which confer resistance toseveral structurally unrelated classes of herbicides in plants, planttissues and seeds. The patent discloses herbicides which normallyinhibit AHAS activity.

Herbicide resistant AHAS large subunit genes have also been rationallydesigned. WO 96/33270, U.S. Pat. Nos. 5,853,973 and 5,928,937 disclosestructure-based modeling methods for the preparation of AHAS variants,including those that exhibit selectively increased resistance toherbicides such as imidazolines and AHAS inhibiting herbicides. Thisdocument discloses isolated DNAs encoding such variants, vectorscontaining these DNAs, methods for producing the variant polypeptides,and herbicide resistant plants containing specific AHAS gene mutations.

The prokaryotic AHAS enzymes exist as two distinct, but physicallyassociated, protein subunits. In prokaryotes, the two polypeptides, a“large subunit” and a “small subunit,” are expressed from separategenes. Three major AHAS enzymes, designated I, II and III, all havinglarge and small subunits, have been identified in enteric bacteria. Inprokaryotes, the AHAS enzyme has been shown to be a regulatory enzyme inthe branched amino acid biosynthetic pathway (Miflin, B. J. Arch.Biochm. Biophys. 146:542-550, 1971), and only the large subunit has beenobserved as having catalytic activity. From studies of AHAS enzymes frommicrobial systems, two roles have been described for the smallsubunit: 1) the small subunit is involved in the allosteric feedbackinhibition of the catalytic large subunit when in the presence ofisoleucine, leucine or valine or combinations thereof; and 2) the smallsubunit enhances the activity of the large subunit in the absence ofisoleucine, leucine or valine. The small subunit has also been shown toincrease the stability of the active conformation of the large subunit(Weinstock et al. J. Bacteriol. 174:5560-5566, 1992). The expression ofthe small subunit can also increase the expression of the large subunitas seen for AHAS I from E. coli (Weinstock et al. J. Bacteriol.174:5560-5566, 1992).

In these microbial systems, the large subunit alone in vitro exhibits abasal level of activity that cannot be feedback-inhibited by the aminoacids isoleucine, leucine or valine. When the small subunit is added tothe same reaction mixture containing the large subunit, the specificactivity of the large subunit increases.

The large AHAS subunit protein has been identified in plants andisolated and used to transform plants. An AHAS mutant allele isotype ofthe AHAS3 large subunit protein, having the tryptophan at position 557replaced with leucine has been found in a Brassica napus cell line(Hattori et al. Mol. & Gen. Genet. 246: 419-425, 1995). The mutantprotein product of this gene confers sulfonylurea, imidazolinone andtriazolopyridine resistance to the cell line. This mutant allele, whenexpressed in transgenic plants, also confers resistance to theseherbicides.

An AHAS herbicide-resistant, double-mutant allele of the large subunitof Arabidopsis thaliana has also been identified (Planta 196:64-68,1995). The gene, csr1-4, encodes an AHAS enzyme with altered kineticswhich is resistant to chlorsulfuron, imazapyr, and triazolopyrimidine.The csr1-4 gene when expressed in plants affects the growth of theplants in response to added L-valine and L-leucine.

Until recently, there was no direct evidence that a small subunitprotein of AHAS existed in eukaryotic organisms. Recently, other groups,through the use of Expressed Sequence Tags (ESTs), have identifiedsequences homologous to the microbial AHAS small subunit genes in aeukaryote, the plant Arabidopsis. They showed that a randomly isolatedArabidopsis cDNA sequence had sequence homology with AHAS small subunitsequences from microbial systems. Since then, ESTs from small subunitgenes have been described from other eukaryotes such as yeast and redalgae (Duggleby 1997, Gene 190:245). Duggleby discloses three ESTsequences, two from Arabidopsis and one from rice, that have homology toknown prokaryotic small subunit gene sequences.

More recently, WO 98/37206 discloses the use of an ALS small subunitcDNA sequence from Nicotiana plumbaginifolia for screening herbicideswhich inhibit the holoenzyme. Until the present invention, however, thecomplete genomic sequence of a eukaryotic AHAS small subunit proteingene had not been determined, nor had a eukaryotic AHAS small subunitprotein been produced or isolated from Arabidopsis.

SUMMARY OF THE INVENTION

The present invention provides DNA sequences encoding a biologicallyfunctional eukaryotic AHAS small subunit protein and functional variantsthereof. In accordance with the invention, the Arabidopsis AHAS smallsubunit gene has been cloned and sequenced. Expression vectorscontaining DNA sequences encoding a eukaryotic AHAS small subunitprotein are provided for transforming plants. Expression vectors arealso provided which contain genes encoding both large and small subunitsof an AHAS protein or proteins. The vectors may be used in methods toproduce transgenic plants of interest, such as dicot and monocotcrop-plants, including wheat, barley, rice, sugarcane, cotton, corn,soybean, sugar beet, canola and the like. The transgenic plants soproduced will possess an elevated level of tolerance to certainherbicides, such as imidazolinones.

The invention also relates to methods for constructing DNA vectorsincluding plasmids containing the AHAS small subunit protein genes. Thevectors of the invention are suitable for transforming a broad range ofplants and may also be engineered to contain a DNA sequence encoding anAHAS large subunit protein.

Vectors containing the complementary eukaryotic small subunit proteingene, for example, the gene derived from Arabidopsis or maize, can beused to transform an imidazolinone-tolerant plant to enhance herbicideresistance by a secondary mechanism.

In a specific embodiment of the invention, expression vectors areprovided which contain DNA sequences encoding the large and small AHASsubunit proteins and the promoters for the large and small subunits ofAHAS as coordinately regulated expression systems in plants.

For certain monocot crops, it may be preferred to use a monocot AHASsmall subunit gene and promoter such as those derived from rice ormaize. These genes are useful for applications involving the developmentof transgenic monocot plants which exhibit herbicide resistance toimidazolinones or other AHAS-inhibiting herbicides.

In one embodiment, the invention relates to a method for creatingtransgenic crop plants that exhibit high level tolerance or resistanceto imidazolinone herbicides. The method comprises introducing a DNAconstruct, such as a plasmid vector containing an herbicide resistantmutant of the AHAS large subunit gene and an AHAS small subunit gene,into a plant that is normally sensitive or partially resistant toimidazolinones. Once the vector is introduced into plant tissue, thevector uses the endogenous mechanisms of the plant to express the largeand small subunit proteins. The increased production of exogenous largeand small subunits of the AHAS enzyme confers enhanced imidazolinoneresistance to the plant. This increased imidazolinone resistance resultsfrom an increase in catalytic activity, stability, resistance todegradation or resistance to inhibition of the large subunit protein inthe presence of increased amounts of small subunit protein in the plant.

In a preferred embodiment, the large and small subunit genes of the AHASenzyme are present on a single plasmid which integrates as one into thegenome of the transformed plants, and segregates as a single locus foreasier breeding of herbicide resistant crops.

In another embodiment of the invention, the DNA construct or vectorcomprises a herbicide-resistant large AHAS subunit gene and a small AHASsubunit protein gene fused into a single gene, operably linked to andexpressed from a single promoter.

The small subunit protein of AHAS produced by the present vectors mayalso be used as a new target site for herbicides or used in combinationwith the large subunit to screen for putative inhibitors of the largesubunit.

The invention also relates to methods of utilizing the small subunit DNAsequences as screening tools to identify mutations of the AHAS enzymewhich confer herbicide resistance in plants. In this aspect of theinvention, organisms coexpressing the large and small subunit of AHASare screened for mutations which confer resistance to herbicides inplants. The mutant gene products are isolated and tested in vivo for theeffects of herbicides including imidazolinones. Then, mutant herbicideresistant forms are isolated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the pUC19 plasmid plant expression vector constructcontaining an Arabidopsis AHAS small subunit genomic DNA sequences ofthe invention.

FIG. 2 depicts an Arabidopsis AHAS small subunit gene map.

FIG. 3 depicts a plasmid expression vector, pHUWE82 of the invention,which contains the Arabidopsis AHAS small subunit gene without the firstthree codons.

FIG. 4 depicts a plasmid expression vector, pHUWE83 of the invention,which contains the Arabidopsis AHAS small subunit gene minus thenucleotide sequence coding for the first 98 amino acids.

FIG. 5 is a bar graph showing the in vitro activity and stability of thelarge AHAS subunit protein of the Arabidopsis AHAS enzyme in thepresence of Phosphate Buffered Saline (MTPBS) and dithiothreitol (DTT).

FIG. 6 is a bar graph showing the in vitro activity of the large subunitprotein of the Arabidopsis AHAS enzyme in the presence of PhosphateBuffered Saline (MTPBS).

FIG. 7 is a graph showing the in vitro activity of the large subunitprotein of the Arabidopsis AHAS enzyme in the presence of increasingamounts of bovine serum albumin (BSA).

FIG. 8 is a graph showing the activity in vitro of the wild type largesubunit protein of the AHAS enzyme in increasing amounts of theArabidopsis small subunit protein.

FIG. 9 is a graph showing the in vitro activity of an herbicideresistant mutant (Met124 His) large subunit Arabidopsis AHAS protein inthe presence of the Arabidopsis AHAS small subunit protein.

FIG. 10 is a plant transformation vector, pHUWE67 of the invention whichcontains a 5.6 kb DNA fragment containing the AHAS small subunit genomicDNA.

FIGS. 11A-11E illustrate the plant transformation (expression) vectorsof the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the cloning and sequencing of an AHASsmall subunit protein gene of Arabidopsis. SEQ ID NO.:1 in the SequenceListing contains the nucleotide sequence of the Arabidopsis AHAS smallsubunit cDNA. The corresponding amino acid sequence of the encoded AHASsmall subunit polypeptide is shown in SEQ ID NO:2 in the SequenceListing. The genomic DNA sequence of the Arabidopsis AHAS small subunitgene is shown in SEQ ID NO:3 of the Sequence Listing.

The invention also relates to an isolated DNA sequence encoding aeukaryotic acetohydroxy-acid synthase, AHAS small subunit protein.Specifically, the invention relates to a DNA sequence which encodes aplant AHAS small subunit protein, which can be obtained from adicotyledonous plant such as Arabidopsis, or a monocotyledonous plantsuch as rice or maize.

The cloned Arabidopsis AHAS small subunit gene sequences can be used inDNA vector constructs to transform crop plants which are normallysensitive or partially resistant to herbicides such as imidazolinone.The transgenic plants obtained following transformation show increasedresistance to AHAS-inhibiting herbicides such as imidazolinone.

The expression vector systems of the present invention can be used undersuitable conditions to transform virtually any plant cell. Transformedcells can be regenerated into whole plants so that when a gene isexpressed in intact plants, it imparts herbicide resistance to thetransgenic plant.

The DNA sequence encoding the AHAS small subunit may be used in vectorsto transform plants so the plants created have enhanced resistant toherbicides, particularly imidazolinones. The DNA sequence encoding theAHAS small subunit protein may be used in vectors alone or incombination with a DNA sequence encoding the large subunit of the AHASenzyme in conferring herbicide resistance in plants.

The invention also relates to a plant expression vector comprising aeukaryotic promoter and a DNA sequence encoding a eukaryotic AHAS smallsubunit protein. The eukaryotic promoter for use in the expressionvector should be a high level expression plant promoter, such as theArabidopsis AHAS small subunit promoter. The AHAS small subunit genesequences preferably used in the expression vector is a DNA sequencewhich encodes the Arabidopsis small subunit protein.

In another embodiment, the plant expression vector comprises a promoterfor the large subunit of eukaryotic AHAS protein; a DNA sequenceencoding the large subunit of a eukaryotic AHAS protein; a promoter forthe small subunit of the AHAS protein; and a DNA sequence encoding for asmall subunit of a eukaryotic AHAS protein.

In yet another embodiment, the plant expression vector for expressing aheterologous AHAS gene in a plant, comprises a plant promoter and a DNAsequence encoding a fusion protein comprising a large subunit and asmall subunit of a eukaryotic AHAS protein.

In one embodiment, the plant expression vector comprises the ArabidopsisAHAS small subunit promoter and a DNA sequence encoding an Arabidopsissmall subunit AHAS protein.

In another embodiment, the plant expression vector comprises in series apromoter expressible in a plant cell; a DNA sequence encoding a transitAHAS large subunit polypeptide; a DNA sequence encoding a wild type,mature AHAS large subunit protein or variant; a DNA sequence encoding alinker polypeptide transcript; a DNA sequence encoding a matureeukaryotic AHAS small subunit protein; and a plant terminator sequence.In this embodiment, the promoter, the DNA sequence encoding the transitAHAS large subunit protein and the DNA sequence encoding the mature AHASlarge subunit protein which are preferably derived from a dicotyledonousplant, particularly from Arabidopsis. Alternatively, the plantexpression vector may comprise a promoter, a DNA sequence encoding thetransit AHAS large subunit protein and a DNA sequence encoding themature AHAS large subunit protein are derived from a monocotyledonousplant such as maize.

In another embodiment, the plant expression vector comprises a promotersuitable for expression in plants, a DNA sequence encoding a fusionprotein comprising a large subunit and a small subunit of a eukaryoticAHAS protein.

In another embodiment, the plant expression vector comprises DNAsequences for enhancing gene expression, such as introns and leadersequences. In this aspect of the invention, the plant expression vectorcomprises a DNA sequence for regulating AHAS gene expression. The intronsequences may also be a heterologous intron sequence from an intron suchas the maize Adh1 intron and the first intron from the shrunkent-1locus. In addition, the DNA sequences for enhancing gene expression maybe a leader sequence such as the W-sequence from the Tobacco Mosaicvirus.

The invention also relates to an isolated eukaryotic AHAS small subunitprotein. The AHAS small subunit protein has the amino acid sequencecorresponding to SEQ ID NO: 2 in the Sequence Listing. This protein canbe purified from, for example, Arabidopsis and may be used incompositions. Also, the gene can be used to express the Arabidopsissmall subunit protein in a microbe such as E. coli and purified fromextracts of E. coli.

The invention also relates to a method for creating a transgenic plantwhich is resistant to herbicides, comprising transforming a plant with aplant expression vector comprising a DNA sequence encoding a eukaryoticAHAS small subunit protein.

The invention also relates to a method for imparting herbicideresistance to a plant cell, comprising co-transforming the plant cellwith a first plant expression vector comprising a first plantexpressible promoter, and a DNA sequence encoding the large subunit ofthe AHAS protein and a second plant expression vector comprising asecond plant expressible promoter and a DNA sequence encoding the smallsubunit of an eukaryotic AHAS protein.

The invention further relates to a method for enhancing the herbicideresistance of a transgenic plant which expresses a gene encoding an AHASlarge subunit protein or a mutant or variant thereof, comprisingtransforming the transgenic plant with a DNA sequence encoding aeukaryotic small subunit AHAS protein or a mutant or variant thereof.

The invention also relates to a method for enhancing the herbicideresistance in the progeny plants of a plant, which comprises somaticallyor sexually crossing the plant with a transgenic plant whose geneticcomplement comprises a sequence encoding a herbicide-resistant mutant ofthe large subunit of a eukaryotic AHAS protein, and a DNA sequenceencoding the small subunit of an eukaryotic AHAS protein; and selectingfor those progeny plants which exhibit herbicide resistance.

The invention also relates to the transgenic plants and progeny producedby the methods of the invention, which plants exhibit elevatedresistance to imidazolinone and other herbicides.

The invention also relates to a transgenic plant whose geneticcomplement comprises a plant expressible gene comprising a promoter forexpression in plants, a DNA sequence encoding a fusion proteincomprising a large subunit and a small subunit of a eukaryotic AHASprotein and a terminator sequence which functions in plant cells.

The invention also relates to a method for identifying mutations in theplant AHAS genes which confer resistance to herbicides, comprisingexposing an organism to a herbicide compound, which organism possesses aheterologous vector comprising an AHAS small subunit protein gene. Inanother aspect of this embodiment, the heterologous vector may comprisethe AHAS large and the small subunit genes. This method is also usefulas a screening system for testing the effects of herbicides on mutantforms of the AHAS enzyme.

The invention also relates to a method for identifying mutations in theplant AHAS gene(s) which alters the allosteric feedback inhibitioncharacteristics of the enzyme. Mutations that alter the feedbackcharacteristics of the enzyme, in either the AHAS large or small subunitgenes are used to alter amino acid levels in plants, particularly of thebranched chain amino acids. The method comprises: transforming amicrobial strain which is deficient in AHAS enzyme activity with aplasmid expression vector comprising a mutant plant AHAS small subunitgene. A suitable microbial strain which lacks AHAS activity is E. coliMI262. The mutant AHAS large and small subunit genes can be generatedrandomly or rationally designed from protein structural models usingmethods previously described (Ott et al. J. Mol. Biol. 263: 359-368,1996). Once the microbial strain is transformed, they are screened inminimal medium in the presence of one or two, but not three branchedchain amino acids, and then the microbial strains which grow in theminimal medium are identified.

The vectors containing the AHAS small subunit gene can be incorporatedinto plant or bacterial cells using conventional recombinant DNAtechnology. Plants are grown from transformed plant cells and secondgeneration plants can be obtained from the seeds of the transgenicplants. Alternatively, the vectors for transforming plants can berecombinant plant viral vectors containing an expressible AHAS smallsubunit protein gene. In this embodiment, the viral vectors are capableof systemically infecting the target crop plants and capable ofexpressing the AHAS small subunit protein in the host plant withoutdisrupting the genome of the host.

The invention also relates to the AHAS small subunit gene promoter DNAsequences. In this aspect of the invention, AHAS small subunit promotersequences can be used to express heterologous polypeptides.Alternatively, the AHAS small and large subunit promoters can also beused as a coordinately regulated gene system to express heterologousmulti-subunit proteins or to overexpress a single gene.

Identification, Cloning and Sequencing of the AHAS Small Subunit ProteinGene

The EST sequence of the putative Arabidopsis thaliana small subunitprotein, designated P_(—)12197 in the GenBank, was used to clone thecomplete AHAS small subunit gene. Synthetic polymerase chain reaction(PCR) primers were specifically designed to correspond to the putativeAHAS small subunit DNA sequences of Arabidopsis corresponding to theAHAS small subunit EST sequences in deposit in the GenBank. The primerswere synthesized using standard techniques (U.S. Pat. No. 4,683,202;Sambrook et al. Molecular Cloning 2^(nd) Ed., Cold Spring Harbor).

Reverse Transcriptase (RT)-PCR was performed on total RNA isolated fromArabidopsis. Primers designed from the EST sequence were able to amplifyan Arabidopsis cDNA fragment. This fragment was cloned into anInvitrogen TA vector (Invitrogen Cat. No. K2000-01) using standardtechniques. This clone was named pDGR102 and corresponded to a 450base-pair fragment containing a portion of the EST sequence.

The same PCR primers were also used to amplify a fragment from anArabidopsis λ:yes cDNA library. This confirmed that an AHAS smallsubunit gene was present in the library. Using a sense strand primerspecific within pDGR102, and a reverse primer that hybridized to theλ:yes phagemid vector, a fragment that represented the 3′ half of thesmall subunit gene was amplified by PCR utilizing the Arabidopsis totalcDNA library as a template source. This product of approximately 800bases was cloned into the Invitrogen TA vector (Invitrogen, Cat. No.K2000-01) and named clone pDGR106 Clone pDGR106 was also sequenced, andits translated amino acid sequence confirmed that the fragmentrepresented the 3′ half of the small subunit gene by homology to knownprokaryotic small subunit gene sequences. This fragment contained thestop codon and 3′ flanking poly A tail.

The PCR fragment contained in pDGR102 and pDGR106 were found torepresent a 5′ region and the 3′ half, respectively, of the smallsubunit gene, and their DNA sequences overlaped by approximately 188 bp.A unique Ssp I restriction enzyme site located in the overlap region wasused to cleave and ligate the fragment together to reconstruct a nearlyfull-length AHAS small subunit gene (a portion of the N-terminal genewas still missing). The resulting clone was labeled pDGR115.

5′ Rapid Amplification of cDNA Ends (5′ RACE, GIBCO/BRL Cat. No.18374-058) was used to complete sequencing of the 5′ end of theArabidopsis AHAS small subunit gene. Primers designed from the pDGR115sequence were used to clone and extend the sequence to the 5′ end of thesmall subunit gene. Total RNA extracted from Arabidopsis seedling wereused as template. The sequence was extended 650 base pairs and aputative start codon for the N-terminal methionine residue wasidentified. The established full length sequence was used to generate afull length cDNA clone.

A genomic clone to the Arabidopsis small subunit gene was obtained byscreening a Clonetech Arabidopsis genomic lambda library. To screen thelibrary, a 380 bp probe was generated by PCR amplification of a 5′region of the small subunit cDNA gene sequences. The PCR product wasobtained by using the primers; 5′-CAGAGATCATGTGGCTAGTTGA-3′ (SEQ ID NO:4 in the Sequence Listing) and 5′-GAGCGTCGAGAATACGATGTAC-3′ (SEQ ID NO:5 in the Sequence Listing). The 380 base pair PCR product was clonedinto the Invitrogen TA cloning vector. To label the probe the PCR insertwas cut out by Eco RI and labeled with α-³²P dCTP by random priming.Screening of the library was performed on nylon membranes byconventional methods. A lambda phage hybridizing to the probe wasidentified and isolated. The lambda phage DNA was extracted and digestedwith Sal I and the fragments were cloned into pUC19. A primer specificto the small subunit cDNA sequence was used in sequencing reactions withthe various cloned Sal I genomic fragment in order to identify the clonecontaining the small subunit gene. A clone containing AHAS small subunitsequence within a 5.6 kb Sal I fragment was identified and it isillustrated within the pMSg6 plasmid in FIG. 1. The promoter region, thetransit sequence, the mature coding sequence of the small subunit gene,the introns, and the translational terminator were identified bysequencing 4.9 kb of the genomic fragment.

Through comparison of the genomic and cDNA sequences the start codon forthe N-terminal methionine was identified. The cDNA for the small subunitgene codes for a polypeptide of 491 amino acids.

FIG. 2 is a map of the genomic DNA sequences of the Arabidopsis AHASsmall subunit gene. As shown in FIG. 2, and referring to SEQ ID NO: 3 inthe Sequence Listing, this gene contains a promoter which extends fromnucleotide number 1 to nucleotide 757. The start codon for the genecorresponds to nucleotides 758-760. The gene contains 11 introns and 12exons. Exon 1 extends from nucleotide 758 to nucleotide 1006. Intron 1extends from nucleotide 1007 to nucleotide 1084. Exon 2 extends fromnucleotide 1085 to nucleotide 1300. Intron 2 extends from nucleotide1301 to nucleotide 1455. Exon 3 extends from nucleotide 1456 tonucleotide 1534. Intron 3 extends from nucleotide 1535 to nucleotide1659. Exon 4 extends from nucleotide 1660 to nucleotide 1731. Intron 4extends from nucleotide 1732 to nucleotide 2236. Exon 5 extends fromnucleotide 2237 to nucleotide 2320. Intron 5 extends from nucleotide2321 to nucleotide 2486. Exon 6 extends from nucleotide 2487 tonucleotide 2640. Intron 6 extends from nucleotide 2641 to nucleotide2910. Exon 7 extends from nucleotide 2911 to nucleotide 2998. Intron 7extends from nucleotide 2999 to nucleotide 3284. Exon 8 extends fromnucleotide 3285 to nucleotide 3389. Intron 8 extends from nucleotide3390 to nucleotide 3470. Exon 9 extends from nucleotide 3471 tonucleotide 3592. Intron 9 extends from nucleotide 3593 to nucleotide3891. Exon 10 extends from nucleotide 3892 to nucleotide 4042. Intron 10extends from nucleotide 4043 to nucleotide 4285. Exon 11 extends fromnucleotide 4286 to nucleotide 4351. Intron 11 extends from nucleotide4352 to nucleotide 4647. Exon 12 extends from nucleotide 4648 to thestop codon at nucleotide 4737. The transcriptional terminator is locatedin the DNA segment between the stop codon at nucleotide 4737 andnucleotide 4895.

The amino acid sequence of the Arabidopsis AHAS small subunit proteinencoded by the DNA sequence as described above, has high homology toamino acid sequences of AHAS small subunit proteins from prokaryoticorganisms. Homology is particularly high in conserved regions of AHASsmall subunit sequences in prokaryotes. As an example, the ArabidopsisAHAS small subunit gene sequence of the present invention had 42.5%sequence identity to that of the Bacillus subtilis small subunit gene.This indicated that the genomic clone DNA sequences was that of theArabidopsis AHAS small subunit.

FIG. 2 also shows the location of two Genbank EST sequences withaccession numbers P_(—)12197 and P_(—)21856. Both EST sequences hadpreviously been identified to have homology to microbial AHAS smallsubunit genes, suggesting there were two isozymes in Arabidopsis. TheEST sequence from P_(—)12197 was used to clone the AHAS small subunitcDNA and genomic clones of the invention. Analysis of the completed AHASsequences indicated the gene codes for two repetitive amino acidsequences with homology to known AHAS small subunits. The AHAS genesequences are ordered in tandem within the single polypeptide. Aftercomparing the AHAS small subunit gene sequences with the original twoESTs, it was determined that the two ESTs are part of the same gene,each corresponding to similar regions within each repeated sequence.

Some specific Materials and Methods used for cloning the genomic smallsubunit gene. Arabidopsis genomic library—Arabidopsis genomic librarywas bought from Clontech.

Construction of Plasmids Containing the AHAS Small Subunit Gene

DNA molecules containing the AHAS small subunit gene comprising thenucleotide sequence in SEQ ID NO:1 or SEQ ID NO:3 in the SequenceListing, or a functional variant thereof, can be inserted into asuitable heterologous expression vector system in proper orientation andcorrect reading frame. Numerous vector systems, such as plasmids,bacteriophage viruses and other modified viruses, can be used inpracticing the invention. Suitable plasmid vectors include, but are notlimited to, pBR322, pUC8, pUC9, pUC18, pUC19, pBI122, pKC37, pKC101 andTA cloning vectors. Viral vectors such as λgt10, λgt11 and Charon 4 canalso be used.

Construction of F1, F2, F3, pHUWE82, and pHUWE83 Plasmid ExpressionVectors

In the present invention, AHAS small subunit cDNA sequences wereinserted into a pGEX-2T or pGEX-4T-2 E. coli expression vector obtainedfrom Pharmacia. Five different DNA fragments containing the AHAS smallsubunit gene sequences were cloned into the pGEX-2T or pGEX-4T-2expression vectors. These clones were designated F1, F2, F3, pHUWE82,and pHUWE83 all differing in the amount of coding sequence containedwithin the expression vector. Plasmids F1, F2 and F3 contain the AHASsmall subunit cDNA in pGEX-4T-2 E. coli expression vector and aredescribed in more detail in Example 2 below. The AHAS small subunit cDNAin plasmids pHUWE82 and pHUWE83 was cloned in pGEX-2T E. coli expressionvectors. The pHUWE82 vector contained a near full length ArabidopsisAHAS small subunit gene (without the first 3 amino acids). pHUWE83 wasengineered to express the small subunit gene without the putativetransit sequence (without the first 98 amino acids). A map of theplasmids pHUWE82 and pHUWE83 are shown in FIGS. 3 and 4, respectively.The versions of the small subunit gene are expressed in E. coli as aglutathione transferase/AHAS small subunit fusion protein. Afteraffinity purification of the fusion protein the respective proteins arecleaved by thrombin. Due to the incorporation of the five amino acidthrombin cleavage site, i.e., Leu-Val-Pro-Arg-Gly-Ser-(SEQ ID NO:6 inthe Sequence Listing), and the location of protease cleavage, anadditional glycine and serine residue is maintained on the N-terminal ofthe small subunit protein. The resulting AHAS small subunit protein frompHUWE82 has the N-terminal sequence Gly-Ser-Ile-Ser-Val-Ser (SEQ ID NO:7in the Sequence Listing; the first 3 amino acids Met-Ala-Ala were notincorporated into the vector), and the protein from pHUWE83 has theN-terminal sequence Gly-Ser-Met-Ile-Asn-Arg (SEQ ID NO:8 in the SequenceListing; the first 98 amino acids were not incorporated into thevector). In both pHUWE82 and pHUWE83 the N-terminal sequence amino acidsGly-Ser- are remnants of the thrombin cleavage site.

Construction of Plant Transformation/Expression Vectors

Numerous plant transformation vectors and methods for transformingplants are available (An, G. et al. Plant Physiol., 81:301-305, 1986;Fry, J., et al. Plant Cell Rep. 6:321-325, 1987; Block, M. Theor. Appl.Genet. 76:767-774, 1988; Hinchee, et al. Stadler. Genet. Symp.203212.203-212, 1990; Cousins, et al. Aust. J Plant Physiol. 18:481-494,1991; Chee, P. P. and Slightom, J. L. Gene. 118:255-260, 1992; Christou,et al. Trends. Biotechnol. 10:239-246, 1992; D'Halluin, et al.,Bio/Technol. 10:309-314. 1992; Dhir, et al. Plant Physiol. 99:81-88,1992; Casas et al. Proc. Nat. Acad. Sci. USA 90:11212-11216, 1993;Christou, P. In Vitro Cell. Dev. Biol.-Plant; 29P: 119-124, 1993;Davies, et al. Plant Cell Rep. 12:180-183, 1993; Dong, J. Z. andMchughen, A. Plant Sci. 91:139-148, 1993; Franklin, C. I. and Trieu, T.N. Plant. Physiol. 102:167, 1993; Golovkin, et al. Plant Sci. 90:41-52,1993; Guo Chin Sci. Bull. 38:2072-2078. 1993; Asano, et al. Plant CellRep. 13, 1994; Ayeres N. M. and Park, W. D. Crit. Rev. Plant.Sci.13:219-239, 1994; Barcelo, et al. Plant. J. 5:583-592, 1994; Becker,et al. Plant. J. 5:299-307, 1994; Borkowska et al. Acta. Physiol. Plant.16:225-230, 1994; Christou, P. Agro. Food Ind. Hi Tech. 5:17-27, 1994;Eapen et al. Plant Cell Rep. 13:582-586, 1994; Hartman, et al.Bid-Technology 12: 919923, 1994; Ritala, et al. Plant. Mol. Biol.24:317-325, 1994; Wan, Y. C. and Lemaux, P. G. Plant Physiol. 104:3748,1994; Weeks, et al. J. Cell Biochem: 104, 1994). The AHAS small subunitDNA sequences are inserted into any of the vectors using standardtechniques. The selection of the vector depends on the preferredtransformation technique and the target plant species to be transformed.In a preferred embodiment, the AHAS small subunit gene can be clonedbehind a high level expressing plant promoter, and this construct canthen be introduced into a plant that is already transgenic or a plant tobe transformed with an herbicide resistant mutant allele of the largesubunit protein. In this manner, the effectiveness of the herbicideresistance gene may be enhanced by stabilization or activation of thelarge subunit protein. This method may be applied to any plant species;however, it is most beneficial when applied to important crops.

Methodologies for constructing plant expression cassettes andintroducing foreign DNA into plants is generally described in the art.For example, foreign DNA can be introduced into plants, usingtumor-inducing (Ti) plasmid vectors. Other methods utilized for foreignDNA delivery involve the use of PEG mediated protoplast transformation,electroporation, microinjection whiskers, and biolistics ormicroprojectile bombardment for direct DNA uptake. Such methods areknown in the art. (U.S. Pat. No. 5,405,765 to Vasil et al.; Bilang etal. (1991) Gene 100: 247-250; Scheid et al., (1991) Mol. Gen. Genet,228: 104-112; Guerche et al., (1987) Plant Science 52: 111-116; Neuhauseet al., (1987) Theor. Appl. Genet. 75: 30-36; Klein et al., (1987)Nature 327: 70-73; Howell et al., (1980) Science 208: 1265; Horsch etal., (1985) Science 227: 1229-1231; DeBlock et al., (1989) PlantPhysiology 91: 694-701; Methods for Plant Molecular Biology (Weissbachand Weissbach, eds.) Academic Press, Inc. (1988) and Methods in PlantMolecular Biology (Schuler and Zielinski, eds.) Academic Press, Inc.(1989). The method of transformation depends upon the plant cell to betransformed, stability of vectors used, expression level of geneproducts and other parameters.

The components of the expression cassette may be modified to increaseexpression of the inserted gene. For example, truncated sequences,nucleotide substitutions or other modifications may be employed. DNAsequences for enhancing gene expression may also be used in the plantexpression vectors. These include the introns of the maize Adh1, intron1gene (Callis et al. Genes and Development 1:1183 -1200, 1987), andleader sequences, (W-sequence) from the Tobacco Mosaic virus (TMV),Maize Chlorotic Mottle Virus and Alfalfa Mosaic Virus (Gallie et al.Nucleic Acid Res. 15:8693-8711, 1987 and Skuzeski et al. Plant Molec.Biol. 15:65-79, 1990). The first intron from the shrunkent-1 locus ofmaize, has been shown to increase expression of genes in chimeric geneconstructs. U.S. Pat. Nos. 5,424,412 and 5,593,874 disclose the use ofspecific introns in gene expression constructs, and Gallie et al. (PlantPhysiol. 106:929-939, 1994) also have shown that introns are useful forregulating gene expression on a tissue specific basis. To furtherenhance or to optimize AHAS small subunit gene expression, the plantexpression vectors of the invention may also contain DNA sequencescontaining matrix attachment regions (MARs). Plant cells transformedwith such modified expression systems, then, may exhibit overexpressionor constitutive expression of the AHAS small subunit gene.

To obtain efficient expression of the AHAS small subunit gene and othergenes of the present invention, a promoter must be present in theexpression vector. Depending upon the host cell system utilized, any oneof a number of suitable promoters can be used. Suitable promoters shouldbe high level expression plant promoters which include ubiquitin, nospromoter, the small subunit ribulose bisphosphate carboxylase genepromoter, the small subunit chlorophyll A/B binding polypeptidepromoter, the 35S promoter of cauliflower mosaic virus, the AHAS largeand small subunit promoters, (OCS)3 MAS and promoters isolated fromplants and plant viruses. See C. E. Vallejos, et al., “Localization inthe Tomato Genome of DNA Restriction Fragments Containing SequencesHomologous to the _(R)RNA (45S), the major chlorophyll _(A/B)bindingPolypeptide and the Ribulose Bisphosphate Carboxylase Genes,” Genetics112: 93-105 (1986). Another promoter suitable for transforming plants isthe actin promoter. A preferred promoter of the invention is the AHASsmall subunit promoter for use with the AHAS small subunit genesequences. The expression vector can then be used to transform a plantcell. Other plant tissues suitable for transformation include leaftissues, root tissues, meristems, cultured plant cells such as calluses,and protoplasts.

Use of the AHAS small subunit gene promoter: In a preferred embodiment,the promoter to be used to transcribe the AHAS small subunit gene shouldbe a strong plant promoter. The native promoter of the AHAS smallsubunit gene in plants may be used for expressing the AHAS small subunitprotein gene in transgenic plants. The small subunit gene promoter canalso be used in vectors to express heterologous genes. The AHAS smallsubunit promoter can also be used in vectors in conjunction with thepromoter of the AHAS large subunit gene. These promoters, which woulddrive the transcription of the two genes coding for two subunits of asingle multimeric protein, may be utilized as coordinately regulatedpromoters. For example, both promoters may be upregulated simultaneouslyat a specific time or in a specific tissue such as meristems. Theadvantage of two different, but simultaneously, active promoters is thatthey may not be susceptible to co-suppression. Co-suppression can occurwhen two genes of similar sequence are present within a transformedorganism. Co-suppression causes the same level of silencing ofexpression of the genes.

Moreover, a transformation vector containing two genes regulated withthe same promoter sequence can undergo recombination between likesequences, thereby inactivating one or both genes. Use of differentpromoters that are co-regulated may allow for expression of two geneswithout problems of recombination, and facilitates the expression ofmultimeric proteins.

The promoter of the AHAS small subunit gene can be used for additionalpurposes. First, the large and the small subunit genes code forpolypeptides that work in concert and in physical contact with eachother, the two genes may be coordinately regulated in expression. Havingboth the large and small subunit promoter may enable the expression ofother multimeric proteins, or overexpression of the same gene from twodifferent promoters. This is advantageous since expressing two geneswith the same promoter may cause problems due to recombination ofhomologous promoter sequences. If two different promoters are used,genes for multimeric proteins may not be expressed at the same time orin the same tissue. Coordinately regulated but heterologous promoterswould overcome these problems.

Secondly, having both the large and small subunit promoter providestools to understand gene regulation. These promoters can be ligated toreporter genes to test for determining the coordination of the level,tissue specificity, and coordination of subunit genes.

Thirdly, it is advantageous to express the small subunit gene on its ownpromoter so that it is expressed at the appropriate time and tissue tohave the most effect in enhancing herbicide resistance.

Lastly, having two promoters that may be coordinately regulated providesus with a tool for analyzing, and isolating regulatory factors that maybe common to each of the promoters. Such factors include transcriptionfactors that regulate expression from both AHAS large and small subunitpromoters. The promoter sequences may also have common motifs that maybe involved in coordinate regulation of the two genes. Moreover, therole of introns in the small subunit genomic clone may be involved inco-regulation of the two promoters. The two promoters and the intronsprovide us with tools to elucidate the mechanism of coordinateregulation of promoters.

Use of Introns of AHAS Small Subunit Gene

The genomic clone has several introns which may be used to regulate geneexpression. Introns have been shown to regulate gene expression. Forexample the maize Adh1 intron 1 significantly increases expression ofreporter genes in maize (Callis et al. 1987, Genes & Development1:1183-1200 by Cold Spring Harbor Laboratory ISSN 0890-9369/87). Thefirst intron of the shrunkent-1 locus of maize has also been shown toincrease expression in chimeric gene constructs. U.S. Pat. Nos.5,424,412 and 5,593,874 disclose the use of specific introns forregulating gene expression.

Introns have also been shown to regulate the level of expression on atissues specific basis. Gallie et al. (Plant Physiol. 106:929-939)showed that enhancement of gene expression by introns is dependent oncell type. Therefore, the AHAS small subunit gene introns can be used toexpress genes with particular emphasis in specific tissue types.

It is also advantageous to express the small subunit gene with all ofits introns so that it is expressed at the appropriate time and tissueto have the most effect in enhancing herbicide resistance.

Use of the AHAS Small Subunit Gene in Expression Vectors

Tethered enzyme. In this embodiment of the invention, the AHAS smallsubunit is translationally coupled to the large subunit via a transcriptcoding for a linker polypeptide, such as polyglycine (polyGly). Thelength of the linker polypeptide tether is varied. The positioning ofthe two AHAS subunits with respect to the linker polypeptide tether isthe large subunit transit sequence followed by the large subunit maturecoding sequence, the linker polypeptide transcript, and the smallsubunit mature coding sequence. An alternative positioning involvesswitching the mature coding sequences of the large and small subunitsabout the linker polypeptide transcript with the small subunit transitsequence.

Tethered enzymes to enhance activity and herbicide resistance. It hasbeen shown with the E. coli enzyme that the association between largeand small subunits is loose. It was estimated that in E. coli at aconcentration of 10⁻⁷ M for each subunit, the large subunits are onlyhalf associated as the α₂β₂ active holoenzyme (Sella et al. 1993, J.Bacteriology 175:5339-5343). Greatest activity is achieved in molarexcesses of the AHAS small subunit protein. Since it has been determinedthat the AHAS enzyme is most stable and active when both subunits areassociated (Weinstock et al 1992, J. Bacteriology, 174:5560-5566, Sellaet al. 1993, J. Bacteriology 175:5339-5343) a highly active and stableenzyme may be created by fusing the two subunits into a singlepolypeptide. Tethered polypeptides have been shown to function properly.Gilbert et al. expressed two tethered oligosacharide synthetic enzymesin E. coli to produce an enzyme that was functional, stable in vitro,and soluble (Gilbert et al. Nature Biotechnology 16: 769-772,1998).

Expression of both the large and small subunits of AHAS as a singlepolypeptide from a single gene also has advantages for producingtransgenic herbicide resistant crops. The use of a single gene totransform and breed plants into elite crop lines is easier and moreadvantageous than when two or more genes are used.

Fused enzyme pair. In this aspect of the invention, the AHAS smallsubunit is positioned in the plant vector directly downstream of thelarge subunit under the direction of a single promoter. Alternatively,the small and large subunit genes of AHAS can be separated and put underthe direction of different promoters within a single construct.

Two genes, one construct. In another aspect of the invention, in thisexpression vector, both the large and the small subunit of AHAS areplaced under the control of separate promoters, in a single plasmidconstruct. This enables the expression of both genes as separateentities; however, the tandem would behave in the plant progeny as asingle locus.

Two genes, one promoter. The maize streak virus promoter is abi-functional promoter able to express genes in two direction. Usingthis promoter, gene transcription can be initiated on genes coded onopposite strands in the vector and in opposite directions. Therefore, alarge and a small subunit genes of AHAS can be expressed from a singlepromoter.

Two genes, two constructs. In another approach, two separate vectorconstructs are made, each containing either the large or small subunitunder the direction of different promoters. This approach requires thatthe plant be doubly transformed.

The plant expression vector should also contain a suitable transcriptionterminator sequence downstream from the gene sequences. A variety oftranscriptional terminators are known for use in plant expressioncassettes for correct termination of gene transcription andpolyadenylation of the transcript. Terminators for use in the inventioninclude CaMV 35S terminator, the nopaline synthase terminator, the peabcs terminator, the tml terminator, the AHAS large and small subunitterminators. These terminators can be used in vectors for use in bothmonocotyledon and dicotyledon transformation.

The gene products of the invention may also be targeted to thechloroplasts. This is accomplished by introducing a signal sequencewhich can be fused into the gene and thus into the expression vector.The signal sequence which will correspond to the amino terminal end ofthe gene product (see Comai et al. J. Biol. Chem. 263:15104-15109, 1988)is fused into the upstream 5′ end of the gene. The AHAS small subunitprotein of the invention has been found to possess a signal sequence,and therefore, this signal sequence can be used in the vectors of theinvention. Other signal sequences for use in the invention are known,such as those from the 5′ end of cDNAs from the AHAS large or smallsubunit, CAB protein, the EPSP synthase, the GS2 protein, and the like(Cheng & Jogendorf, J. Biol. Chem. 268:2363-2367, 1993).

Bacteria from the genus Agrobacterium can be utilized to introduceforeign DNA and transform plant cells. Suitable species of suchbacterium include Agrobacterium tumefaciens and Agrobacteriumrhizogenes. Agrobacterium tumefaciens (e.g., strains LBA4404 or EHA105)is particularly useful due to its well-known ability to transformplants.

Another approach to transforming plant cells with a heterologous geneinvolves propelling inert or biologically active particles at planttissues and cells. U.S. Pat. Nos. 4,945,050; 5,036,006; and 5,100,792all to Sanford et al. disclose this technique. In summary, thisprocedure involves propelling inert or biologically active particles atthe cells under conditions effective to penetrate the outer surface ofthe cell in such manner as to incorporate the vectors into the interiorof the cells. When inert particles are utilized, the vector can beintroduced into the cell by coating the particles with the vectorcontaining the desired gene. Alternatively, the target cell can besurrounded by the vector so that the vector is carried into the cell bythe wake of the particle. Other biologically active particles includingdried yeast cells, dried bacteria, or bacteriophages, each containingthe desired DNA, can also be propelled into plant cell tissue. Inaddition, the vectors of the invention can be constructed so that theyare suitable for use in plastid transformation methods using standardtechniques.

The isolated AHAS small subunit gene of the present invention can beutilized to confer herbicide resistance to a wide variety of plant cellsincluding monocots and dicots. Although the gene can be inserted intoany plant cell falling within these broad classes, it is particularlyuseful in crop plant cells, such as those of rice, wheat, barley, rye,corn, carrot, sugarcane, tobacco, bean, pea, soybean, sugar beet, andcanola.

The expression system of the present invention can be used to transformvirtually any crop plant cell under suitable conditions. Transformedcells can be regenerated into whole plants such that the AHAS smallsubunit gene imparts or enhances herbicide resistance to the intacttransgenic plants. As set forth above, the expression system can bemodified so that the herbicide resistance gene is continuously orconstitutively expressed.

Use of the AHAS small subunit gene to enhance herbicide resistance inplants: A plasmid that contains both the genes for the AHAS large andsmall subunit can be constructed. In this manner, the two genes wouldsegregate as a single locus making breeding of herbicide resistant cropseasier. Alternatively, the large and small subunit can be fused into asingle gene expressed from a single promoter. The fusion protein wouldhave elevated levels of activity and herbicide resistance. The largesubunit of AHAS may be of a wild type sequence (if resistance isconferred in the presence of an independent or fused small subunit), ormay be a mutant large subunit that in itself has some level ofresistance to herbicides. The presence of the small subunit will 1)enhance the activity of the large subunit, 2) enhance the herbicideresistance of the large subunit, 3) increase the stability of the enzymewhen expressed in vivo and 4) increase resistance to large subunit todegradation. The small subunit would in this manner elevate theresistance of the plant/crop to the imidazolinone or other herbicides.The elevated resistance would permit the application and/or increase thesafety of weed-controlling rates of herbicide without phytotoxicity tothe transformed plant/crop. Ideally, the resistance conferred wouldelevate resistance to imidazolinone and other classes of herbicideswithout increasing, or by increasing to a lesser degree, resistance toother AHAS inhibiting herbicides such as sulfonylureas,triazolopyrimidines, etc.

Additional Aspects About Enhancing Herbicide Resistance by the Additionof the Small Subunit Gene to Plants Expressing a Herbicide ResistantForm of the Large Subunit Gene.

It has been shown in many cases that mutations in proteins can causeinstability, decreases in activity, and a greater propensity todegradation. Herbicide resistant AHAS genes, particularly those fromplants, generally contain a mutation that confers the resistance toinhibition by the herbicide. This places a greater level of importancein stabilizing, maintaining activity, and resistance to degradation ofthese proteins. The accompaniment of the small subunit gene to the largesubunit gene may assist in these areas of susceptibility.

Subunits of multi-subunit proteins that are present in the absence ornon-stoichiometric levels of the complementary subunits can bepreferentially degraded. This has been shown for the large and smallsubunits of ribulosebisphosphate carboxylase/oxygenase (Spreitzer et al.Proc. Natl. Acad. Sci. USA 82: 5460-5464). If the large subunit gene forAHAS is transformed and expressed in a crop that does not have thecomplementary small subunit, or is expressed at high levels beyond theexpression levels of the small subunit gene, the large subunit proteincould be unstable and preferentially degraded. This could result in alower level of herbicide resistance.

The association of large and small subunits appears to be highlyspecific. E. coli has three isozymes of large subunits and threeisozymes of small subunits. Each large subunit isozyme specificallyassociates with only one of the small subunit isozymes, even though allsubunits are expressed in the same organism (Weinstock et al 1992, J.Bacteriology, 174:5560-5566). This specificity suggests that endogenousAHAS small subunit proteins could not stabilize or enhance the activityof an introduced AHAS large subunit if the large subunit gene is derivedfrom a different organism or isozyme pair from that of the smallsubunit. This places an importance, for purposes of herbicideresistance, in introducing both the large and small subunit genes fromthe same organism and isozyme pair. The expression of a small subunitgene may ameliorate these problems.

The AHAS small subunit gene in combination with the AHAS large subunitgene can also be used as a marker for selecting transformant plant cellsand tissues. Any gene of interest can be incorporated in vectorscontaining the AHAS large and small subunit genes. The vectors can beintroduced into plant cells or tissues that are succeptible toAHAS-inhibiting herbicides. The transformants containing these vectorscan be selected in the presence of herbicides using standard techniques.

EXAMPLE 1

DNA and Lambda DNA isolation: DNA isolation was carried out using QIAgenSpin Miniprep Kit (50) (QIAgen Cat. No. 27104) and standard proceduresas provided by the manufacturer. For Lambda DNA isolation, the QIAgenLambda Midi Kit (25) (QIAgen Cat. No. 12543) was used following themanufacture's protocol. In TA Cloning, the Invitrogen Original TACloning Kit (Invitrogen Cat. No. K2000-01) was used and standardprotocols were followed.

Subcloning: Lambda DNA was digested with the appropriate restrictionenzyme and mixed with pUC19 which was digested with the same restrictionenzyme. After phenol extraction, the insert was ligated with pUC19 byadding 1 μl DNA ligase (4 units/mL) and incubate at 17° C. overnight.

5′ RACE: The 5′ RACE used in the experiments was 5′ RACE System forRapid Amplification of cDNA Ends and was obtained from GIBCO/BRL (Cat.No. 18374-058). The reactions were carried out following standardprocedures provided by the manufacturer.

Screening library: The Clonotech Arabidopsis lambda genomic library wasplated at a density of 30,000 plaques/150 mm plate as described in theprotocol supplied by the manufacturer. Amersham Nucleic Acid TransferMembranes Hybond™-N+ (DISC: 0.137 m DIA, Removal Rating: 0.45 um) wereused. The nylon transfer membranes were carefully placed onto the platesurface, and membranes and agar were marked using a sterile needle. Thefirst membrane was removed after 3 minutes and a duplicate membrane wasplaced on the plate surface and removed after 8 minutes. The membraneswere placed, colony side up, on a pad of absorbent filter paper soakedin denaturing buffer (0.5N NaOH, 1.5 N NaCl) for 5 minutes, then eachmembrane was placed, colony side up, on a pad of absorbent filter papersoaked in neutralizing solution (1M Tris-HCl, 1.5N NaCl) for 5 minutes.The membranes were washed briefly in 2×SSC, and transferred to dryfilter paper. The sample was fixed to the membranes by UV crosslinkingand vacuum-baked at 80° C. for 1 hour. Then the membranes wereprehybridized in a buffer containing 50% formamide, 2×SSC, 5×Denhardt'ssolution, 1% sodium dodecyl sulfate (SDS), 0.05 mg/ml denatured salmonsperm DNA, and 0.05% NaPPi at 42° C. for 2 hour. DNA was digested withrestriction enzyme and fractionated on a 1% agarose gel. The DNAfragment containing the AHAS small subunit gene was purified usingQIAquick Gel Extraction Kit (50) (QIAgen Cat. No. 28704). The GIBCO/BRLLife Technologies Random Primers DNA labeling System (Cat. No.18187-013) was used to label the DNA with the following modification.125 ng of probe DNA was dissolved in 55 μL of distilled water in amicrocentrifuge tube and denatured by heating for 5 minutes in a boilingwater bath, and immediately cooled on ice. Then, dATP, dGTP, dTTP,[α-³²P]dCTP and Klenow enzyme were added to the denatured DNA, and themixture was incubated at 25° C. for an hour. One volume of formamide wasadded to the mixture and the reaction was heated at 65° C. for 30minutes. The reaction containing the labeled DNA was added toprehybridization solution and the membranes were hybridized at 42° C.for 20 hours with slow shaking. After hybridization, the membranes werewashed twice with 0.4×SSC buffer containing 0.1% SDS at room temperaturefor 10 minutes, followed by a single wash in 0.2×SSC buffer containing0.1% SDS at 65° C. for 30 minutes. The membranes were exposed to X-rayfilm overnight. Plaques containing DNA which hybridized to the DNA probeon duplicate membranes produced a positive result, and these plaqueswere isolated. The procedure was duplicated until single isolates couldbe collected.

Sequencing: The sequencing reaction was carried out using the ABI PRISMDNA sequencing Kit following the ABI protocol provided. After ethanolprecipitation, the DNA was dissolved in ABI PRISM Template SuppressionReagent and denatured at 90° C. for 5 minutes. Then, the samples wereloaded onto an ABI 310 sequencer.

EXAMPLE 2 Preparation of Plasmid DNA Containing AHAS Large SubunitProtein Genes

The wild type (pAC774) and Met92His mutant (pAC786) AHAS large subunitgenes from Arabidopsis were constructed into the E. coli expressionvector pGEX-4T-2 from Pharmacia. The genes were constructed to express aglutathione transferase/AHAS large subunit fusion protein to aid inpurification, similarly as described for plasmids pHUWE82 and pHUWE83 asdescribed above. A five amino acid protease cleavage site was encoded atthe junction of the two proteins so that they could be cleaved apartafter purification. Three vector constructs containing the cDNAsequences of the Arabidopsis AHAS small subunit gene were made using thepGEX4T-2 expression vector. These were designated F1, F2 and F3, allthree differing in the amount of peptide sequence contained within thegene. The N-terminal amino acid sequence of the peptide encoded by theAHAS cDNA of the F1 plasmid is Gly-Ser-Pro-Lys-Ile-Ala-Leu-Arg- (SEQ IDNO: 9 in the Sequence Listing). The N-terminal amino acid sequence ofthe peptide encoded by the AHAS cDNA of plasmid F2 isGly-Ser-Leu-Asp-Ala-His-Trp- (SEQ ID NO: 10 in the Sequence Listing).The N-terminal amino acid sequence of the peptide encoded by the AHAScDNA of the F3 plasmid is Gly-Ser-Val-Glu-Pro-Phe-Phe- (SEQ ID NO: 11 inthe Sequence Listing). The N-terminal Gly-Ser- for all three peptidesare remnants of the thrombin cleavage.

Expression of the Arabidopsis Large and Small Subunits Proteins of AHAS

DH5α-competent cells (Gibco BRL) were transformed with large subunitplasmids pAC774 and pAC786, as well as small subunit plasmids F1, F2,and F3. Cells were thawed on ice. 1 μl of a 1:5 dilution of the plasmidDNA was added to 75 μl of cells, which then sat on ice for 30 minutes.Cells were heat shocked in a 42° C. water bath for 90 seconds and thenput on ice for two minutes. 800 μl of Luria-Bertani medium (LB) wasadded to each tube containing transformed cells which were then grownfor 1 hour in a 37° C. shaker. Cells were centrifuged for 2 minutes andexcess medium was aspirated. The cell pellet was resuspended in 100 μlof LB and plated on LB containing 100 μg of carbenicillin overnight at37° C. Single colonies were inoculated into 50 ml of LB mediumcontaining 375 μg/ml carbenicillin and grown overnight in a 37 ° C.shaker. 700 μl aliquots were taken and added to 300 μl of 50% glycerol,and were then frozen in liquid nitrogen and kept at −80° C. for cellstocks.

Purification of the Large Subunit AHAS Gene

An overnight 50 ml culture of transformed E. coli harboring the largesubunit gene in the pGEX-2T expression vector was inoculated into 1liter of 2×YT with 2% glucose, 375 μg/ml Carbenicillin. Cells were grownfor 5 hours in a 37° C. shaker/incubator and then induced with 0.1 mMIPTG and then placed in a 30° C. shaker for another 2.5 hours. Cellswere harvested by centrifugation in a JA10 rotor at 8000 rpm at 4° C.for 10 minutes. Cells were stored as a pellet at −20° C. untilpurification.

The cell pellet from 1 liter of cell culture was resuspended in 10 ml ofMTPBS (150 mM NaCl, 16 mM Na₂HPO₄, 4 mM NaH₂PO₄), pH 7.3 (Smith andJohnson Gene 67: 31-40, 1998) and 100 μg/ml of lysozyme was added. Thesuspension was adjusted to 5 mM dithiothreitol. Triton X-100 was addedto a final concentration of 1% by addition of a 20% Triton X-100 inMTPBS solution. The cells were shaken gently at 30° C. for 15 minutesand were then cooled to 4° C. on ice and sonicated for 8-10 secondsusing a microtip probe, duty cycle 70%, output control at maximum forthe microtip pulser. The sonicate was centrifuged two times in a J20rotor, 17,000 rpm, 4° C., 10 minutes, to remove insoluble material.Lysate was added to 150 mg (dry weight) of glutathione agarose(equilibrated and hydrated in MTPBS) and inverted for 30 minutes at 4°C. Agarose was settled by centrifugation at 500 rpm for 5 minutes andwas washed with ice cold MTPBS by repeated centrifugation cycles untilthe A₂₈₀ of the wash matched that of MTPBS. Agarose was transferred toan appropriate column for elution. Fusion proteins were eluted with 50mM Tris-HCl, 5 mM reduced glutathione into 1 ml fractions. The A₂₈₀ ofeach fraction was checked for protein content and appropriate fractions(A₂₈₀>0.100) were pooled. To the pooled sample were added 5 units ofbovine thrombin per ml of protein solution. The sample was dialyzedagainst MTPBS, 3 mM dithiothreitol for 15 hours at room temperature toallow proteolytic cleavage of the fusion protein and removal of Tris-HCland reduced glutathione. Dialyzed sample was passed twice more throughequilibrated glutathione agarose to remove the cleaved GST protein andto remove uncut fusion protein. Purified samples were stored at 4° C.with or without 0.02% sodium azide.

Purification of Small Subunit Protein of AHAS

Transformed DH5 cells were cultured and harvested in a manner similar tothat used for collecting cells expressing the large subunit of AHAS. Thecell pellet from 1 liter of culture was resuspended in 10 ml of STE (150mM NaCl, 10mM Tris-HC1 pH 8.0, 1 mM EDTA), and then 100 μg/ml oflysozyme was added. This was put on ice for 15 minutes. Dithiothreitolwas added to 5mM and N-lauroylsarcosine was added to a finalconcentration of 1.5% from a 10% stock in STE. The cells were vortexedfor 10 seconds. The lysate was sonicated on ice for 8-10 seconds using amicrotip probe, duty cycle 70%, output control at maximum for themicrotip pulser. The sonicate was centrifuged two times in a JA20 rotorat 4° C. at 17,000 rpm for 10 minutes, to remove insoluble material. Thesupernatant was added to 200 mg (dry weight) of glutathione agarose(equilibrated and hydrated in MTPBS) and inverted for 20 minutes at 4°C. The agarose was settled by centrifugation at 500 rpm for 5 minutes,and the supernatant was aspirated. The agarose was washed with ice coldMTPBS by repeated centrifugation cycles until the A₂₈₀ of the wash wasequal to that of MTPBS. The agarose was transferred to an appropriatecolumn for elution. Fusion proteins were eluted with 50 mM Tris-HCL pH8.0, 10 mM reduced glutathione, 5 mM dithiothreitol, and 2%N-octylglucoside, and 1 ml fractions were collected. The absorbance at280 nm of each fraction was checked, and appropriate fractions(A₂₈₀>0.100) were pooled. The sample was dialyzed for 15 hours againstMTPBS, 3 mM dithiothreitol, at 4° C. to remove reduced glutathione,Tris-HCl, and N-octylglucoside. SDS was added to 0.005% from a 1% stocksolution in H₂O. Five units of bovine thrombin were added per ml ofprotein solution, and the sample was shaken gently at room temperaturefor 30-45 minutes to allow proteolytic cleavage of the fusion protein.The sample was then immediately passed through an EtractiGel-D detergentaffinity column (Pierce) to remove SDS. The cut sample was stored at 4°C. or passed twice through re-equilibrated glutathione agarose to removeGST and uncut fusion protein. The purified sample was stored at 4° C.

Determination of Protein Concentration

An aliquot of protein solution was adjusted to 5% trichloroacetic acidand put on ice for 20 minutes to allow precipitation of protein. Thealiquot was then centrifuged for 10 minutes at high speed and 4° C. topellet the precipitate. The pellet was resuspended in an equivalentvolume of 3% (w/v) sodium carbonate, 0.1N NaOH. A Pierce BCA proteinassay was used to determine the concentration of the protein solution.Bovine serum albumin standards were prepared in 3% sodium carbonate,0.1N NaOH.

AHAS assays: AHAS assays were performed with slight modification asdescribed by Singh et al. {Singh et al., 1988}. AHAS activity wasassayed in 1×AHAS assay buffer (50 mM HEPES pH 7.0, 100 mM pyruvate, 10mM MgCl₂, 1mM TPP, and 50 μM FAD). A final concentration of 1×assaybuffer was obtained either by dilution of enzyme extracted in 2×assaybuffer or addition of enzyme to make a final concentration 1X AHAS assaybuffer. All assays containing imazethapyr and associated controlscontained a final concentration of 5% DMSO due to addition ofimazethapyr to assay mixtures as a 50% DMSO solution. Assays wereperformed in a final volume of 250 μL at 37° C. in microtiter plates.

EXAMPLE 3

Effects of Dithiothreitol. The effect of dithiothreitol (DTT) inPhosphate Buffered Saline (MTPBS) on AHAS large subunit enzyme activitywas measured. Experiments were carried out with or without 3 mM DTT inthe purified protein solution of large subunit. The assays wereconducted as above. The results are presented in FIGS. 5 and 6. As canbe seen in FIG. 5 when compared to FIG. 6, the catalytic activity of theAHAS large subunit is enhanced dramatically by DTT. Moreover, in theabsence of DTT, large subunit activity degraded over the 4 day period(FIG. 6). The Arabidopsis large subunit was found to be very stable inthe presence of 3 mM DTT over a period of 1 month when stored at 4° C.

Other sulfhydryl reductants were tested, but DTT appeared to bothstabilize and activate the enzyme better than reduced glutathione andβ-mercaptoethanol. Due to the activation of the enzyme by DTT allexperiments addressing activation of the AHAS large subunit enzyme bythe small subunit enzyme were performed either in the absence of DTT orother sulfhydryl reducing agents or at a constant concentration of 3 mMDTT.

Effects of Bovine Serum Albumin. To determine whether the enhancement ofthe large subunit was specific to the small subunit, another nonspecificcontrol which tested the effects of bovine serum albumin was run. To afixed amount of large subunit an increasing amount of a 0.25 mg/ml BSAsolution was added. The results are shown in FIG. 7. As seen in FIG. 7,the addition of BSA to the test sample caused a slight, but notsignificant, and a plateaued increase in the catalytic activity of thelarge subunit protein of AHAS.

EXAMPLE 4

The following experiments used the small subunit and large subunitproteins purified as described in Example 2 above. The plasmid F1containing the AHAS small subunit gene from Arabidopsis was expressed inE. coli and partially purified. The protein concentration of the samplewas determined and aliquots of increasing concentration were added to aconstant amount of purified Arabidopsis large subunit.

FIG. 8 shows the activation of the wild type Arabidopsis AHAS largesubunit by addition of the Arabidopsis small subunit protein. AHASassays were carried out as described above and the results shown in FIG.8 indicate that small subunit protein enhances the level of enzymaticactivity of the catalytic large subunit. The activation of the largesubunit protein of AHAS is shown for both the wild type large subunitand a herbicide-resistant mutant of the large subunit.

In another experiment, a herbicide-resistant mutant of the large subunitenzyme (substitution mutation at position 124, methionine substituted byhistidine) was used. The results shown in FIG. 9 demonstrate that theenzymatic activity of a herbicide-resistant form of the large subunit isalso enhanced.

EXAMPLE 5

The plant transformation vector, pHUWE67 illustrated in FIG. 10containing the Arabidopsis AHAS small subunit genomic DNA wasconstructed as follows. The pUC 19 vector shown in FIG. 1 containing the5.6 kb genomic fragment of the Arabidopsis AHAS small subunit gene(pMSg6) was cut with Sal I. The 5.6 kb fragment containing the entireArabidopsis AHAS small subunit gene (see FIG. 2), including the promoterand introns was separated from the vector by agarose gelelectrophoresis. The fragment was cut out of the agarose gel andpurified using the QIAquick Gel Extraction Kit (Cat. No. 28706)following the procedures provided by the manufacturer. The Agrobacteriumbased transformation vector, pBIN19, was cut with Sal I. The vector waspurified by phenol:chloroform extractions. The purified vector wasdephosphorylated by treatment with calf intestinal alkaline phosphataseand re-extracted with phenol:chloroform. The vector and genomic insertwere ligated and the ligation mix containing the construct was used totransform E. coli strain DH5α. E. coli was selected on kanamycin platesand plasmids were extracted from transformed E. coli. The vectorconstruct was verified by generation of a PCR product and sequencing ofthe product using the sequencing procedures described in Example 1. Thevector designated pHUWE67 (FIG. 10), thus contains a 5.6 kb fragmentcomprising the AHAS genomic DNA containing the AHAS promoter, an OpenReading Frame (ORF) and 3′-terminator fused with the pBIN19 plasmid.This vector construct is used for Agrobacterium based transformation ofplants using standard techniques.

The plant transformation vectors illustrated in FIGS. 11A-11E aresimilarly constructed as vector pHUWE67 above, following standardcloning procedures. In FIGS. 11A-11E, the vector may comprise an AHASsmall subunit cDNA, fragment, genomic fragment or mutant. In FIG. 11Bthe vector further comprises an AHAS small subunit promoteroperably-linked upstream of the gene insert. In this embodiment, theArabidopsis small subunit gene includes the terminator.

In FIG. 11C the vector construct contains the AHAS small and largesubunit genes in tandem, with the large subunit gene downstream from thesmall subunit gene. Both genes are regulated by the AHAS small subunitpromoter which is located upstream from the gene inserts. The AHAS largesubunit gene is a herbicide resistant mutant allele.

In FIG. 11D the plant expression vector contains the large and smallsubunit genes under the control of their own promoters. Transcriptiontermination signal is provided by the AHAS small subunit terminator. TheAHAS large subunit gene confers resistant to herbicides such asimidazolinone.

The vector in FIG. 11E is similar to the vector represented in FIG. 11C.In this vector, however, the AHAS large and small subunit genes arereversed in position in the construct. The AHAS large subunit gene inthis example is upstream of the small subunit gene. The AHAS largesubunit gene is a herbicide resistant mutant allele.

Based on the techniques of the present invention and following stringentDNA hybridization techniques, homologous AHAS small subunit genesequences can be obtained from a variety of plant species, such as rice,maize, wheat, barley, and the like. Therefore, these AHAS small subunitgene sequences are also useful in the present vectors and methods fortransforming plants.

11 1 1673 DNA Arabidopsis sp. CDS (42)..(1514) Mature Peptide 1gtcttcttca gtagcaaaaa accttcggct tcgtctcgtc a atg gcg gcc att tct 56 MetAla Ala Ile Ser 1 5 gta agt tct tca cca tct att cgc tgc ttg aga tcg gcatgt tcc gat 104 Val Ser Ser Ser Pro Ser Ile Arg Cys Leu Arg Ser Ala CysSer Asp 10 15 20 tct tct cct gct ctt gta tcc tcg acg cgt gta tcg ttc ccggcg aag 152 Ser Ser Pro Ala Leu Val Ser Ser Thr Arg Val Ser Phe Pro AlaLys 25 30 35 att tca tat ctc tcc ggt ata tct tcg cac cgt ggc gat gaa atgggt 200 Ile Ser Tyr Leu Ser Gly Ile Ser Ser His Arg Gly Asp Glu Met Gly40 45 50 aag aga atg gaa gga ttc gtt aga agc gtc gat ggg aag atc tct gat248 Lys Arg Met Glu Gly Phe Val Arg Ser Val Asp Gly Lys Ile Ser Asp 5560 65 gcg tct ttc tcc gaa gct tca tct gcg act cca aaa tcg aag gtg agg296 Ala Ser Phe Ser Glu Ala Ser Ser Ala Thr Pro Lys Ser Lys Val Arg 7075 80 85 aag cac aca att tca gta ttt gtt gga gac gaa agc gga atg att aat344 Lys His Thr Ile Ser Val Phe Val Gly Asp Glu Ser Gly Met Ile Asn 9095 100 agg att gca gga gtg ttt gca agg aga gga tac aat att gag agt ctt392 Arg Ile Ala Gly Val Phe Ala Arg Arg Gly Tyr Asn Ile Glu Ser Leu 105110 115 gct gtt ggt ctg aac aga gac aag gct cta ttc acc ata gtt gtc tgt440 Ala Val Gly Leu Asn Arg Asp Lys Ala Leu Phe Thr Ile Val Val Cys 120125 130 gga act gaa agg gta ctt cag cag gtc atc gag caa ctc cag aag ctc488 Gly Thr Glu Arg Val Leu Gln Gln Val Ile Glu Gln Leu Gln Lys Leu 135140 145 gtt aat gtt cta aag gtt gaa gat atc tca agt gag ccg caa gtg gag536 Val Asn Val Leu Lys Val Glu Asp Ile Ser Ser Glu Pro Gln Val Glu 150155 160 165 cgt gag ctg atg ctt gta aaa gtg aat gca cat cca gaa tcc agggca 584 Arg Glu Leu Met Leu Val Lys Val Asn Ala His Pro Glu Ser Arg Ala170 175 180 gag atc atg tgg cta gtt gac aca ttc aga gca aga gtt gta gatata 632 Glu Ile Met Trp Leu Val Asp Thr Phe Arg Ala Arg Val Val Asp Ile185 190 195 gcg gaa cat gca ttg act atc gag gta act gga gat cct gga aaaatg 680 Ala Glu His Ala Leu Thr Ile Glu Val Thr Gly Asp Pro Gly Lys Met200 205 210 att gct gta gaa aga aat ttg aaa aag ttt cag atc aga gag attgta 728 Ile Ala Val Glu Arg Asn Leu Lys Lys Phe Gln Ile Arg Glu Ile Val215 220 225 agg aca gga aag ata gca ctg aga agg gaa aag atg ggt gca actgct 776 Arg Thr Gly Lys Ile Ala Leu Arg Arg Glu Lys Met Gly Ala Thr Ala230 235 240 245 cca ttt tgg cga ttt tca gca gca tcc tat cca gat ctc aaggag caa 824 Pro Phe Trp Arg Phe Ser Ala Ala Ser Tyr Pro Asp Leu Lys GluGln 250 255 260 gcg cct gtt agt gtt ctt cga agt agc aaa aaa gga gcc attgtc cct 872 Ala Pro Val Ser Val Leu Arg Ser Ser Lys Lys Gly Ala Ile ValPro 265 270 275 caa aag gaa aca tca gca ggg gga gat gtt tat ccc gtt gagcca ttt 920 Gln Lys Glu Thr Ser Ala Gly Gly Asp Val Tyr Pro Val Glu ProPhe 280 285 290 ttt gac ccc aag gta cat cgt att ctc gac gct cac tgg ggactt ctc 968 Phe Asp Pro Lys Val His Arg Ile Leu Asp Ala His Trp Gly LeuLeu 295 300 305 act gac gaa gat acg agt gga cta cgg tcg cat act cta tcattg ctt 1016 Thr Asp Glu Asp Thr Ser Gly Leu Arg Ser His Thr Leu Ser LeuLeu 310 315 320 325 gta aat gat att cca gga gtt ctt aat att gtg act ggtgtt ttc gct 1064 Val Asn Asp Ile Pro Gly Val Leu Asn Ile Val Thr Gly ValPhe Ala 330 335 340 cga agg gga tac aat atc cag agc ttg gcc gta gga catgct gaa acc 1112 Arg Arg Gly Tyr Asn Ile Gln Ser Leu Ala Val Gly His AlaGlu Thr 345 350 355 aag ggc att tca cgc att aca aca gtt ata cct gca acagat gaa tcg 1160 Lys Gly Ile Ser Arg Ile Thr Thr Val Ile Pro Ala Thr AspGlu Ser 360 365 370 gtc agc aaa ttg gtg cag caa ctt tac aaa ctc gta gatgtg cat gag 1208 Val Ser Lys Leu Val Gln Gln Leu Tyr Lys Leu Val Asp ValHis Glu 375 380 385 gtc cat gat ctt act cat ttg cca ttt tct gaa aga gaactg atg ctg 1256 Val His Asp Leu Thr His Leu Pro Phe Ser Glu Arg Glu LeuMet Leu 390 395 400 405 att aag att gcc gtg aac gct gct gct aga aga gatgtc ctg gac att 1304 Ile Lys Ile Ala Val Asn Ala Ala Ala Arg Arg Asp ValLeu Asp Ile 410 415 420 gct agt att ttc agg gct aaa gct gtt gac gta tctgat cac aca att 1352 Ala Ser Ile Phe Arg Ala Lys Ala Val Asp Val Ser AspHis Thr Ile 425 430 435 act ttg cag ctt act ggg gat cta gac aag atg gttgca ctg caa agg 1400 Thr Leu Gln Leu Thr Gly Asp Leu Asp Lys Met Val AlaLeu Gln Arg 440 445 450 tta ttg gag ccc tat ggt ata tgt gag gtt gca agaacc ggt cgt gtg 1448 Leu Leu Glu Pro Tyr Gly Ile Cys Glu Val Ala Arg ThrGly Arg Val 455 460 465 gca ttg gct cgt gaa tcg gga gtg gac tcc aag tacctt cgt gga tac 1496 Ala Leu Ala Arg Glu Ser Gly Val Asp Ser Lys Tyr LeuArg Gly Tyr 470 475 480 485 tcc ttt ctt tta aca ggc taaaccgttgcagagtgcat ccatcgaaca 1544 Ser Phe Leu Leu Thr Gly 490 tcagaaactttggaaggtaa aagtttcatt acacagtcta tgaacctcaa agacagacag 1604 agagactgcgtcgatatatg tttgtgactt tgtttatgaa acaattagct gattttgggc 1664 ttcatttcg1673 2 491 PRT Arabidopsis sp. 2 Met Ala Ala Ile Ser Val Ser Ser Ser ProSer Ile Arg Cys Leu Arg 1 5 10 15 Ser Ala Cys Ser Asp Ser Ser Pro AlaLeu Val Ser Ser Thr Arg Val 20 25 30 Ser Phe Pro Ala Lys Ile Ser Tyr LeuSer Gly Ile Ser Ser His Arg 35 40 45 Gly Asp Glu Met Gly Lys Arg Met GluGly Phe Val Arg Ser Val Asp 50 55 60 Gly Lys Ile Ser Asp Ala Ser Phe SerGlu Ala Ser Ser Ala Thr Pro 65 70 75 80 Lys Ser Lys Val Arg Lys His ThrIle Ser Val Phe Val Gly Asp Glu 85 90 95 Ser Gly Met Ile Asn Arg Ile AlaGly Val Phe Ala Arg Arg Gly Tyr 100 105 110 Asn Ile Glu Ser Leu Ala ValGly Leu Asn Arg Asp Lys Ala Leu Phe 115 120 125 Thr Ile Val Val Cys GlyThr Glu Arg Val Leu Gln Gln Val Ile Glu 130 135 140 Gln Leu Gln Lys LeuVal Asn Val Leu Lys Val Glu Asp Ile Ser Ser 145 150 155 160 Glu Pro GlnVal Glu Arg Glu Leu Met Leu Val Lys Val Asn Ala His 165 170 175 Pro GluSer Arg Ala Glu Ile Met Trp Leu Val Asp Thr Phe Arg Ala 180 185 190 ArgVal Val Asp Ile Ala Glu His Ala Leu Thr Ile Glu Val Thr Gly 195 200 205Asp Pro Gly Lys Met Ile Ala Val Glu Arg Asn Leu Lys Lys Phe Gln 210 215220 Ile Arg Glu Ile Val Arg Thr Gly Lys Ile Ala Leu Arg Arg Glu Lys 225230 235 240 Met Gly Ala Thr Ala Pro Phe Trp Arg Phe Ser Ala Ala Ser TyrPro 245 250 255 Asp Leu Lys Glu Gln Ala Pro Val Ser Val Leu Arg Ser SerLys Lys 260 265 270 Gly Ala Ile Val Pro Gln Lys Glu Thr Ser Ala Gly GlyAsp Val Tyr 275 280 285 Pro Val Glu Pro Phe Phe Asp Pro Lys Val His ArgIle Leu Asp Ala 290 295 300 His Trp Gly Leu Leu Thr Asp Glu Asp Thr SerGly Leu Arg Ser His 305 310 315 320 Thr Leu Ser Leu Leu Val Asn Asp IlePro Gly Val Leu Asn Ile Val 325 330 335 Thr Gly Val Phe Ala Arg Arg GlyTyr Asn Ile Gln Ser Leu Ala Val 340 345 350 Gly His Ala Glu Thr Lys GlyIle Ser Arg Ile Thr Thr Val Ile Pro 355 360 365 Ala Thr Asp Glu Ser ValSer Lys Leu Val Gln Gln Leu Tyr Lys Leu 370 375 380 Val Asp Val His GluVal His Asp Leu Thr His Leu Pro Phe Ser Glu 385 390 395 400 Arg Glu LeuMet Leu Ile Lys Ile Ala Val Asn Ala Ala Ala Arg Arg 405 410 415 Asp ValLeu Asp Ile Ala Ser Ile Phe Arg Ala Lys Ala Val Asp Val 420 425 430 SerAsp His Thr Ile Thr Leu Gln Leu Thr Gly Asp Leu Asp Lys Met 435 440 445Val Ala Leu Gln Arg Leu Leu Glu Pro Tyr Gly Ile Cys Glu Val Ala 450 455460 Arg Thr Gly Arg Val Ala Leu Ala Arg Glu Ser Gly Val Asp Ser Lys 465470 475 480 Tyr Leu Arg Gly Tyr Ser Phe Leu Leu Thr Gly 485 490 3 4895DNA Arabidopsis sp. promoter (1)..(757) Promoter Region 3 tcgcatattgttccggcgag gatcatgtga agcttgacgc gtgaattgac gactaagcgt 60 acgacgaagcgatccagttg agaattgtct cgagattcct cgttttagct gtcccactac 120 attcgccatgatttcgaaat ctctttctct tcttctctct ttcgtcttct tctgcgaaaa 180 aatcgaatggataatcacat tttctttttc tcgagaaaat tgatctggtg attatgtgag 240 atccgtctctagcgcgttgc ttatcgagaa ataattaatt ttaatttgac gggtgaagat 300 attattggcgacgtctgttt ccgattgact ttgatttgac ttttcctttc aatcattatt 360 tggcgagtcccgcgtaaata tggactcttc ttgattgtcc cacttttttc ggtggcttta 420 ccggatttaaaatcattttc ttttcctaaa ttatgaattt taccctaaac ttctcataat 480 tacaattagttccgacgaac ccaagatact ttttagcaaa attaggaaaa tagttgactc 540 gaaaaggttgttataacgtg gagctgacgt gttggtctta tctactcgaa gccttttggg 600 cttttcttaaagccattgat ttctaaggtc gtcaacaacc gaaccggacc ggacggtttg 660 accggtctaaccaacatata tacgttcttt ttcnacttgc cgtttcgtcg tcgtcagtct 720 tcttcagtagcaaaaaacct tcggcttcgt ctcgtcaatg gcggccattt ctgtaagttc 780 ttcaccatctattcgctgct tgagatcggc atgttccgat tcttctcctg ctcttgtatc 840 ctcgacgcgtgtatcgttcc cggcgaagat ttcatatctc tccggtatat cttcgcaccg 900 tggcgatgaaatgggtaaga gaatggaagg attcgttaga agcgtcgatg ggaagatctc 960 tgatgcgtctttctccgaag cttcatctgc gactccaaaa tcgaagcgac tgtgaataat 1020 atttgcttaaagtcgtttcc ttttggcctt tgctttgatt gattctttgt gcattaaaat 1080 cagggtgaggaagcacacaa tttcagtatt tgttggagac gaaagcggaa tgattaatag 1140 gattgcaggagtgtttgcaa ggagaggata caatattgag agtcttgctg ttggtctgaa 1200 cagagacaaggctctattca ccatagttgt ctgtggaact gaaagggtac ttcagcaggt 1260 catcgagcaactccagaagc tcgttaatgt tctaaaggtt gttcttttgt tagatcgcac 1320 ttattagtttctgcatgact atagtttcat tcgcaccaac tttacgcatc agccaatttg 1380 cttattcattatttgaagat tagatttgcg atttcctttt ccattctctt cattgacttg 1440 gacatgaattaggttgaaga tatctcaagt gagccgcaag tggagcgtga gctgatgctt 1500 gtaaaagtgaatgcacatcc agaatccagg gcagaggtac tattccttgc ctatgggaaa 1560 ttagagtttactgtacttgc tggttgcttc tgatttaggg cagaggtggt gttagttttc 1620 tctctaaatttgattaagct tctgttttaa tgaattcaca gatcatgtgg ctagttgaca 1680 cattcagagcaagagttgta gatatagcgg aacatgcatt gactatcgag gtacatctac 1740 ttattatgatttgtgttggt cttgatattt gtttcgcact gtagcctgtg ggtttcaaga 1800 cttctgtttgaacatcttac taatcgttgg aagacatcag aaatattatg gagggatcat 1860 tttaacttttatatctatta gttggatttt cgttgccttt tgaaactgat gatgatccac 1920 atgcaggactctattatagg atgtgtatta aagtttattt gaaacttttg gtgcaacttc 1980 ttgaatttaatataacgaga aagttattca acagtgtgct acctttgatt accctatgct 2040 tataatctgtattctgagtt gtattgcctg tgcaaatttc tgtgggaatg ctcagtgttc 2100 acttttgaaagttagagaag cataacctta aatatattgt tctttttacc ttgattatga 2160 gaaagtggagtaaaagaaag ggtgtctctg atttacctat tttagctctt tagtaatcat 2220 ttttaagctattttgcaggt aactggagat cctggaaaaa tgattgctgt agaaagaaat 2280 ttgaaaaagtttcagatcag agagattgta aggacaggaa aggtagtgta tgtttggaat 2340 tactagattttatggctttt gaatatcatc tagtttgtgc tatctaatgt atgtatgtag 2400 tagttacatctttgagtgga cacaaaggca tagatctcag ggactttcac taatttaggg 2460 aaaatggaatgacatttttg gataacagat agcactgaga agggaaaaga tgggtgcaac 2520 tgctccattttggcgatttt cagcagcatc ctatccagat ctcaaggagc aagcgcctgt 2580 tagtgttcttcgaagtagca aaaaaggagc cattgtccct caaaaggaaa catcagcagg 2640 ggtgtgtgcttctctgctcc ttagattgtt taacttcagc ttgaagttcc tcactttcct 2700 ttcaaaaaatttggttgcat aaattatagt aggtttggct atttgataaa gttaaacagc 2760 aactatagatgcctgtgttt ttttccctct atgtggtggc tgcctggaat caacatttga 2820 agcatgcccttttttgtttt tctccctggc tgcactgaag gatttccgag tttgctaatt 2880 tttaaaagttatcttatctt tttaaatgta gggagatgtt tatcccgttg agccattttt 2940 tgaccccaaggtacatcgta ttctcgacgc tcactgggga cttctcactg acgaagatgt 3000 aagagagttctttgctatat atctaacttc gtgccatgaa tttgctaaaa agcaatatga 3060 aaaattcagattgtggtttg cattacacga gttacacttg tttttccatt caagccgtct 3120 ggcataatcaattctgttaa tatagttaca taaatgataa atcaattgag tgttagattt 3180 ggagactgtatgtatttact tacaaagcag acattgaaag agttggggtt ttctttaagc 3240 tatttcgttttatttatcac agttattctt ttttgatctt tcagacgagt ggactacggt 3300 cgcatactctatcattgctt gtaaatgata ttccaggagt tcttaatatt gtgactggtg 3360 ttttcgctcgaaggggatac aatatccagg catagtcctt atctctctca tacacgcaca 3420 cacagtgtgcttaggtttac tgacacactg aaagatctcc tttcttttag agcttggccg 3480 taggacatgctgaaaccaag ggcatttcac gcattacaac agttatacct gcaacagatg 3540 aatcggtcagcaaattggtg cagcaacttt acaaactcgt agatgtgcat gaggtgggat 3600 taccaaaagctactgtcttt cttatatatt taacagtttg aatgtctttg atggccctat 3660 cattcctttgctgtcttaga ccttttggct ttttttaaaa cgtagattag aggaagagtt 3720 tctgctaaatctttctggac tttcctatat catttcctgg tcttgtctgt ttactcgaat 3780 gagacctcttgttccagaaa gtcaaactgt acaggcttga tgaaaataat tctgaacatg 3840 atttgccgcaactttccaag ctgttattaa ctttgtgagg attttctgca ggtccatgat 3900 cttactcatttgccattttc tgaaagagaa ctgatgctga ttaagattgc cgtgaacgct 3960 gctgctagaagagatgtcct ggacattgct agtattttca gggctaaagc tgttgacgta 4020 tctgatcacacaattacttt gcaggtaaaa tacatttctc ataaatggga tttttatgta 4080 gctgttattgcatctcagat gagaaatcct ttcaattgga gatcttcaaa gtttcacgtc 4140 tttccataggtcttcaactt gtttgacata atcagagttc cgtttgaaaa aaatatatga 4200 agctgacttggattttccat cttaatctct tttttttgct tttgtgtttt ggatttgtgt 4260 gctgaaatttgttggctgtg ggtatagctt actggggatc tagacaagat ggttgcactg 4320 caaaggttattggagcccta tggtatatgt gaggtttgtt tcgcaatcta ctttcatctc 4380 ttagtgaatgcataaccccg tgaattctta tttcttataa tgctacccca attgctccgg 4440 ataaagtcccaaaatttagt tgtagtcttt acgacttaga aacagagtag tgaacatcta 4500 actctctggtaaaatcaata accaaagctg gacctagtta catgaatctt cttctggttg 4560 tgtgtagaacaagaataagc ttgacaagcc atgactactt tcagattatg catcgtgttg 4620 acgcttattatgaacaatca atcacacagg ttgcaagaac cggtcgtgtg gcattggctc 4680 gtgaatcgggagtggactcc aagtaccttc gtggatactc ctttctttta acaggctaaa 4740 ccgttgcagagtgcatccat cgaacatcag aaactttgga aggtaaaagt ttcattacac 4800 agtctatgaacctcaaagac agacagagag actgcgtcga tatatgtttg tgactttgtt 4860 tatgaaacaattagctgatt ttgggcttca tttcg 4895 4 22 DNA Artificial SequenceDescription of Artificial SequencePrimer made from cDNA sequence 4cagagatcat gtggctagtt ga 22 5 22 DNA Artificial Sequence Description ofArtificial SequencePrimer made from cDNA sequence 5 gagcgtcgagaatacgatgt ac 22 6 6 PRT Artificial Sequence Description of ArtificialSequenceThrombin cleavage site 6 Leu Val Pro Arg Gly Ser 1 5 7 6 PRTArabidopsis sp. PEPTIDE (1)..(6) N-terminal of AHAS small subunitpeptide of pHUWE82 7 Gly Ser Ile Ser Val Ser 1 5 8 6 PRT Arabidopsis sp.PEPTIDE (1)..(6) N-terminal of AHAS small subunit peptide of pHUWE83 8Gly Ser Met Ile Asn Arg 1 5 9 8 PRT Arabidopsis sp. PEPTIDE (1)..(8)N-terminal sequence of AHAS small subunit peptide of plasmid F1 9 GlySer Pro Lys Ile Ala Leu Arg 1 5 10 7 PRT Arabidopsis sp. PEPTIDE(1)..(7) N-terminal sequence of AHAS small subunit peptide of plasmid F210 Gly Ser Leu Asp Ala His Trp 1 5 11 7 PRT Arabidopsis sp. PEPTIDE(1)..(7) N-terminal of AHAS small subunit peptide from plasmid F3 11 GlySer Val Glu Pro Phe Phe 1 5

We claim:
 1. An isolated DNA molecule encoding a functional eukaryoticacetohydroxy-acid synthase (AHAS) small subunit protein, wherein saidDNA molecule hybridizes to a DNA molecule comprising a sequence havingthe complement of SEQ ID NO: 1 under conditions comprising: (a)hybridization at 42° C. for 20 hours in a solution comprising 50%formamide, 2×SSC, 5×Denhardt's solution, 1% sodium dodecyl sulfate(SDS), 0.05 mg/ml denatured salmon sperm DNA, and 0.05% NaPPi; (b) twowashes at room temperature for 10 minutes in a solution comprising0.4×SSC and 0.1% SDS; and (c) one wash at 65° C. for 30 minutes in asolution composing 0.2×SSC and 0.1% SDS.
 2. The isolated DNA molecule ofclaim 1, wherein said AHAS small subunit protein is a plant AHAS smallsubunit protein.
 3. A plant expression vector comprising a promoterexpressible in a plant cell operably linked to the DNA molecule ofclaim
 1. 4. A transgenic plant whose genetic complement comprises theplant expression vector of claim
 3. 5. A progeny plant of the transgenicplant of claim 4, wherein said progeny plant comprises said plantexpression vector.
 6. A transgenic seed of the transgenic plant of claim4.
 7. An isolated DNA sequence molecule encoding the amino acid sequenceset forth in SEQ ID NO:2.
 8. A plant expression vector comprising apromoter expressible in a plant cell operably linked to the DNA moleculeof claim
 7. 9. A transgenic plant whose genetic complement comprises theplant expression vector of claim
 8. 10. A progeny plant of thetransgenic plant of claim 9, wherein said progeny plant comprises saidplant expression vector.
 11. A transgenic seed of the transgenic plantof claim
 9. 12. A plant expression vector comprising a promoterexpressible in a plant cell operably linked to a DNA molecule comprisingthe sequence set forth in SEQ ID NO:1.
 13. A transgenic plant whosegenetic complement comprises the plant expression vector of claim 12.14. A progeny plant of the transgenic plant of claim 13, wherein saidprogeny plant comprises said plant expression vector.
 15. A transgenicplant whose genetic complement comprises a heterologous promoterexpressible in a plant cell operably linked to an isolated DNA moleculeencoding a small subunit of an Arabidopsis thaliana AHAS protein.
 16. Aprogeny plant of the transgenic plant of claim 15, wherein said progenyplant comprises said heterologous promoter operably linked to said DNAmolecule.
 17. A transgenic plant whose genetic complement comprises aplant expression vector comprising a promoter expressible in a plantcell operably linked to an isolated DNA molecule encoding an Arabidopsisthaliana AHAS small subunit protein.
 18. A progeny plant of thetransgenic plant of claim 17, wherein said progeny plant comprises saidplant expression vector.
 19. A transgenic seed of the transgenic plantof claim
 17. 20. A transgenic plant whose genetic complement comprises aplain expression vector comprising a promoter expressible in a plantcell operably linked to a DNA molecule encoding an Arabidopsis AHASsmall subunit protein, wherein said DNA molecule comprises a sequenceselected from the group consisting of the DNA sequence set fourth in SEQID NO: 1 and the DNA sequence set forth in SEQ ID NO:
 3. 21. A progenyplain of the transgenic plant of claim 20, wherein said progeny plantcomprises said plant expression vector.
 22. A transgenic seed of thetransgenic plain of claim 20.